Target: Inositol-1-monophosphatase NCBI Gene#:17725819 SuhB Protein ID#: 10.1186/1472-6807-7-55 Organism: Mycobacterium tuberculosis Etiologic Risk Group: RG3 Background/Disease Information: Mycobacterium tuberculosis is the bacteria which causes tuberculosis, an infectious disease that mainly affects the lungs [2]. This disease is estimated to be the cause of three million death per year worldwide [1]. The bacteria is spread to individuals by an infected person’s coughs or sneezes. Those at risk include those with HIV, diabetes, cancer, or malnutrition. Many medications exist to treat tuberculosis, though many strains of the disease are resistant to these drugs. Infected individuals must take a cocktail of various types of medications for long periods of time to prevent antibiotic resistance [2]. Essentiality of this protein: The mycobacterial cell wall is composed of various substances based on a phosphatidyl-myo-inositol structure. These substances are extremely important in the cell wall of mycobacterial species. They also greatly contribute to the immunopathogenesis by tuberculosis [1]. Inositol-1-monophosphatase is a critical enzyme in inositol biosynthesis in Mycobacterium tuberculosis SuhB. It synthesizes free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria. Not essential via Brown, et. al. 2007
Complex of proteins?: No, dimerizes in presence of Mg2+ to be active Druggable Target: Yes (0.5 Druggability in TDR Targets)
-- Cost and quantity of substrate reagents and supplier: 40mM pNPP (Sigma): $79.60/1g
Structure Available from PDB -- PDB # 2Q74
Current Inhibitors: Li+ (IC50: 0.9 mM) Zn2+ (IC50:0.5 uM) Mg2+ concentrations higher than 100mM Expression Information: Yes, it has been expressed in E.Coli [1]. Purification Method: His Tag Purification Image of protein : Amino Acid Sequence: 10 20 30 40 50 60 MTRPDNEPAR LRSVAENLAA EAAAFVRGRR AEVFGISRAG DGDGAVRAKS SPTDPVTVVD
Molar Extinction coefficient of your protein at 280 nm wavelength: 45950 TMpred graph Image : *CDS Gene Sequence (paste as text only): *GC% Content for gene: 0.4% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file -that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
[1] Nigou, J.; Dover, L. G.; Besra, G. S., Purification and Biochemical Characterization of Mycobacterium tuberculosis SuhB, an Inositol Monophosphatase Involved in Inositol Biosynthesis†.Biochemistry2002,41 (13), 4392-4398. [2] Mayo Clinic. Tuberculosis. http://www.mayoclinic.com/health/tuberculosis/DS00372/ DSECTION=risk-factors (accessed May 1, 2013)
NCBI Gene#:17725819
SuhB
Protein ID#: 10.1186/1472-6807-7-55
Organism: Mycobacterium tuberculosis
Etiologic Risk Group: RG3
Background/Disease Information:
Mycobacterium tuberculosis is the bacteria which causes tuberculosis, an infectious disease that mainly affects the lungs [2]. This disease is estimated to be the cause of three million death per year worldwide [1]. The bacteria is spread to individuals by an infected person’s coughs or sneezes. Those at risk include those with HIV, diabetes, cancer, or malnutrition. Many medications exist to treat tuberculosis, though many strains of the disease are resistant to these drugs. Infected individuals must take a cocktail of various types of medications for long periods of time to prevent antibiotic resistance [2].
Essentiality of this protein:
The mycobacterial cell wall is composed of various substances based on a phosphatidyl-myo-inositol structure. These substances are extremely important in the cell wall of mycobacterial species. They also greatly contribute to the immunopathogenesis by tuberculosis [1]. Inositol-1-monophosphatase is a critical enzyme in inositol biosynthesis in Mycobacterium tuberculosis SuhB. It synthesizes free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria.
Not essential via Brown, et. al. 2007
Complex of proteins?: No, dimerizes in presence of Mg2+ to be active
Druggable Target: Yes (0.5 Druggability in TDR Targets)
*EC#: 3.1.3.25
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.25&Suchword=&organism%5b%5d=Mycobacterium+tuberculosis&show_tm=0
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.ncbi.nlm.nih.gov/pubmed/11914086
-- Cost and quantity of substrate reagents and supplier: 40mM pNPP (Sigma): $79.60/1g
Structure Available from PDB
-- PDB # 2Q74
Current Inhibitors:
Li+ (IC50: 0.9 mM)
Zn2+ (IC50:0.5 uM)
Mg2+ concentrations higher than 100mM
Expression Information:
Yes, it has been expressed in E.Coli [1].
Purification Method: His Tag Purification
Image of protein :
Amino Acid Sequence:
10 20 30 40 50 60
MTRPDNEPAR LRSVAENLAA EAAAFVRGRR AEVFGISRAG DGDGAVRAKS SPTDPVTVVD
70 80 90 100 110 120
TDTERLLRDR LAQLRPGDPI LGEEGGGPAD VTATPSDRVT WVLDPIDGTV NFVYGIPAYA
130 140 150 160 170 180
VSIGAQVGGI TVAGAVADVA ARTVYSAATG LGAHLTDERG RHVLRCTGVD ELSMALLGTG
190 200 210 220 230 240
FGYSVRCREK QAELLAHVVP LVRDVRRIGS AALDLCMVAA GRLDAYYEHG VQVWDCAAGA
250 260 270 280 290
LIAAEAGARV LLSTPRAGGA GLVVVAAAPG IADELLAALQ RFNGLEPIPD
Number of Amino Acids: 267
Molecular Weight: 28447.1
http://enzyme.expasy.org/EC/3.1.3.25
http://web.expasy.org/cgi-bin/protparam/protparam1?P65164@1-267@
Molar Extinction coefficient of your protein at 280 nm wavelength: 45950
TMpred graph Image :
*CDS Gene Sequence (paste as text only):
*GC% Content for gene: 0.4%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file -that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
[1] Nigou, J.; Dover, L. G.; Besra, G. S., Purification and Biochemical Characterization of Mycobacterium tuberculosis SuhB, an Inositol Monophosphatase Involved in Inositol Biosynthesis†. Biochemistry 2002, 41 (13), 4392-4398.
[2] Mayo Clinic. Tuberculosis. http://www.mayoclinic.com/health/tuberculosis/DS00372/ DSECTION=risk-factors (accessed May 1, 2013)